Collagen modulators for use in photoablation eximer laser keratectomy

ABSTRACT

A method of smoothing irregular corneal surfaces and removing protuberances from corneal surfaces by photoablative eximer laser keratectomy is provided. Collagen compositions for use in making collagen modulators useful in photoablative procedures are described. These compositions are applied to irregular corneal surfaces in sufficient amounts to at least fill in depressions or other irregularities on a corneal surface and are converted into a modulator, as a gel or polymerized film, prior to photoablation. The collagen modulators facilitate the photoablative smoothing of irregular corneal surfaces and protect adjacent corneal tissue from undesired photoablation.

This patent application is a continuation of application Ser. No.08/602,922, filed Feb. 16, 1996, now U.S. Pat. No. 5,861,486 which is acontinuation of Ser. No. 07/942,657, filed Sep. 9, 1992, now U.S. Pat.No. 5,492,135. Each of these prior applications is hereby incorporatedherein by reference, in its entirety.

FIELD AND BACKGROUND OF THE INVENTION

Photoablation eximer laser keratectomy is a ophthalmologic techniquewhich employs an 193 nm excimer laser as a surgical tool to ablate orremove a precise amount of tissue from the anterior corneal surface ofthe eye. In addition to its usual application in correcting refractiveerrors (e.g. myopia, hyperopia and astigmatism) by altering thecurvature of the cornea, this technique has more recently been applied,with success, in removing opacities and irregularities from the anteriorcorneal surface.

The application of eximer lasers in photoablation procedures have beendescribed in the medical literature. See, for example, Sher, N. A.(1991) Arch. Ophthal., Vol. 109, pages 491-498; Zabel, R. W. et al.(1990) Refrac. Corn. Surg., Vol. 6, pages 329-334; Steinert, R. F. etal, ibid, page 352; Gaster, R. N. et al. (1989) Invest. Ophthal. Vis.Sci., Vol. 30, pages 90-98; and Tuft, S. J., ibid, page 1769-1777.Typically, a far ultraviolet argon fluoride laser, emitting at 193 nm,is used in clinical procedures because of its minimal tissueinteraction, ablative efficiency and ease of control of ablation depth.Moreover, irradiation at 193 nm shows less mutagenic potential incomparison to longer ultraviolet wavelengths. The laser, fitted with aseries of apertures of varying diameter and shapes, is preprogrammed todeliver a series of pulses of a given duration and energy fluencesettings. In general, eye movements are minimized during the ablatingprocess and the eye is held with the visual axis fixated under thecenter of the laser beam.

The extent and depth of the ablation depends on a number of variableswhich include aperture, shape and diameter, laser energy fluence(mJ/cm²), duration of irradiation (nanoseconds), pulse rate (Hz) andnumber of pulses. Other factors would include intraoperative epitheliumand corneal stromal drying during effluent removal. Eximer laserablation of the anterior corneal lamellar tissue, in general, leavesbehind a smooth surface that enables reepithelialization, a clearercornea, and an appropriate refractive surface.

In certain situations, modulators are used during the procedure. Asdefined herein, the term “modulator” refers to a substance which, whenapplied to tissue, is capable of absorbing UV irradiation and modulatingthe degree of tissue ablation. Modulators are generally used as adjunctsto promote photoablative smoothing of irregular corneal surfaces and toprotect adjacent corneal tissue where ablation is not desired. Exampleswhich would benefit from the use of modulators include removal ofcorneal scars and opacities, often accompanied with an irregular orrough epithelial surface, due to post-infectious and post-traumaticcauses, including herpes simplex virus, dystrophies (e.g. Salzmanns andReis Buckler's syndrome), recurrent erosions and band keratopathy. Also,several types of corneal pathologies ablate more quickly than others,and this differential ablation may lead to increased irregularity of thecorneal surface following ablation.

A number of photoablation modulators have been reported in theliterature. See, for example, Sher, N. A. (1991), supra; Steinert(1990), supra; Kornmehl, E. W. et al. (1991) Investigative Ophthalmology& Visual Science, Vol. 31(4), Page 245, Abstract no. 1203; and Steinert,R. F. in “Eximer Laser Phototherapeutic Keratectomy: Strategies andRepresentative Cases,” 17th Cornea Research Conference, Sep. 19-21,1991, Eye Research Institute and Massachusetts Eye and Ear Infirmary.Examples of known modulators include viscous aqueous solutions ofmethylcellulose, dextran 70, sodium carboxymethylcellulose andhydroxypropylmethylcellulose 2910 as well as 0.9% saline.

In general, conventional modulators suffer from a number of deficiencieswhich preclude their broader use in photoablation procedures. Forexample, modulators (1) are difficult to apply smoothly on the cornealsurgical bed; (2) are susceptible to drying and rippling from the airflow from the effluent remover; (3) do not adequately absorb at 193 nm;and/or (4) do not adequately promote a smoother ablated corneal surfacerelative to a control situation (no modulator) because the modulatorablates at a different rate than corneal tissue. Accordingly, there is aneed in the art for photoablation modulators which avoid one or more ofthe aforementioned deficiencies.

SUMMARY OF THE INVENTION

Surprisingly, it has now been discovered that certain collagenformulations are useful for preparing collagen modulators useful inphotoablation procedures. These compositions not only fill in variousimperfections on the corneal surface, but also ablate at approximatelythe same rate as the corneal stroma. The collagen modulators promote asmoother ablated surface, relative to a control situation (nomodulator). The disclosed collagen formulations has enormous utility inclinical eximer laser photoablation procedures.

The present invention provides biologically compatible collagensolutions for use in photoablation procedures and methods for itspreparation as well as application in therapeutic and refractive eximerlaser photoablation.

In one embodiment of the invention, a neutralized, acid solubilizedcollagen, which remains in solution at physiological temperatures, isused to prepare a modulator gel coating or film on a corneal surface.These solutions must be extensively dialyzed against EDTA solutionsand/or deionized water to reduce available cations and to preventpremature collagen fibrillogenesis. As the cations are removed, the pHof the collagen solution is increased to between about 6.8 and about 7.5by adjusting the pH of the EDTA solution using 1 N sodium hydroxide. Thecollagen preparation does not undergo typical fibrillogenesis in theabsence of added unbound or free cations.

When applied to a surface of a human cornea prior to photoablationkeratectomy, the collagen formulation uniformly coats and readilyadheres to the surface, filling surface depressions and otherirregularities and provides a smooth surface for subsequentphotoablation. The collagen coating is then instantly converted to afirm gel upon contact with a metal cation-containing solution. Metalcations are supplied in buffer solutions such as phosphate bufferedsaline or sodium chloride solution. The collagen gel readily absorbs UVirradiation, e.g. 193 nm, which is used in eximer laser keratectomy andexhibits ablation properties which resemble the human cornea. Uponcompletion of the photoablation procedure, any remaining residualcollagen gel are readily removed from the corneal surface by dislodgingthe residue with water or physiological buffer solution.

In another embodiment of the invention, chemically modifiedpolymerizable soluble collagen solutions having redox initiators areused in preparing modulator films. When applied to a corneal surface asa coating, the chemically modified collagen adheres to the surface,filling surface depressions and other irregularities. The coating isthen subjected to polymerization conditions such as short wave UV toform a thin modulator film prior to photoablation. The film stronglyadheres to the corneal surface and is physically removed during thephotoablative procedure.

In a further embodiment of the invention, a glutaric anhydride modifiedcollagen, preparable by reacting soluble collagen with glutaricanhydride in an amount ranging between about 20 and about 30 wt. % basedon total collagen, is provided and which undergoes temperature-dependentsol/gel transformation. The glutaric collagen, at a collagenconcentration ranging between about 5 and about 100 mg/ml, melts orliquifies at physiological temperature, e.g. 37° C., to form a viscoussolution. When applied to a corneal surface as a coating at 37° C., theglutaric collagen adheres to the surface, filling surface depressionsand other irregularities. The coating rapidly forms a gel upon coolingto room temperature without any addition of metal cation solution orinduced polymerization. The collagen gel readily absorbs UV irradiation,e.g. 193 nm, which is used in eximer laser keratectomy and exhibitsablation properties which resemble the human cornea. Upon completion ofthe photoablation procedure, any remaining residual collagen gel may bereadily dislodged from the corneal surface with water or physiologicalbuffer solution.

Accordingly, it is an object of the invention to provide a neutralized,acid solubilized collagen solution suitable for use in preparing amodulator for use in photoablation procedures and a method for itspreparation. When such compositions are applied to the corneal surfaceas a coating, it does not undergo fibrillogenesis or gel formationunless contacted with cations.

It is another object of the invention to provide a chemically modifiedpolymerizable collagen solution for use in preparing a collagenmodulator and a method for its preparation. When applied to the cornealsurface as a coating, the collagen solution is polymerized to form afilm which is then removed by the photoablative procedure.

It is a further object of the invention to provide a method forsmoothing an irregular corneal surface having collagen formulations asmodulators by photoablation.

These and other objects of the invention will become apparent in view ofthe detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 (a-e) illustrate the preparation of a neutralized solublecollagen modulator on a irregular corneal surface according to themethod of the present invention. FIGS. (1 f) and (1 g) illustratecross-sections of corneal surfaces having an adherent collagenmodulator.

FIGS. 2 (a-g) illustrate the use of a contact lens to form a smoothcollagen modulator coating on a corneal surface.

FIGS. 3 (a-d) illustrate the present method for removing a protuberancefrom a corneal surface using a collagen modulator.

FIGS. 4 (a-d) illustrate the preparation of a polymerized collagenmodulator film, from a modified collagen solution, on a irregularcorneal surface.

FIGS. 5 (a-g) illustrate the resulting corneal surfaces afterphotoablation without the use of a collagen modulator (FIGS. 5a-c) orwith a collagen modulator (FIGS. 5d-g).

FIG. 6 illustrates that undialyzed, concentrated solubilized collagensolution (3.0 mg/ml), when adjusted to neutral pH (pH 7.2 using 2.5 NNaOH), undergoes fibrillogenesis at 25° C. The absorbance was monitoredat 313 nm for a period of time up to 1 hour.

FIG. 7 illustrates the effect of addition of 0.5 ml of 2.5 M salinesolution to 0.5 ml of neutralized acid soluble collagen solution (3.0mg/ml) at 25° C. The sharp increase in absorbance (at 313 nm) indicatesthat the collagen solution immediately converts into a dense gel uponaddition of saline solution.

FIG. 8a is an SEM photograph which illustrates the effects ofphotoablation on a pig corneal surface without the use of a modulator.

FIG. 8b is an SEM photograph which illustrates the effects ofphotoablation on a pig corneal surface using a collagen gel modulator.

FIG. 9a is an SEM photograph which illustrates the effects ofphotoablation on a surgically induced irregular pig corneal surfacewithout the use of a modulator.

FIG. 9b is an SEM photograph which illustrates the effects ofphotoablation on a surgically induced irregular pig corneal surfaceusing a collagen gel modulator.

FIG. 10a is an SEM photograph which illustrates the effects ofphotoablation on a laser induced irregular pig corneal surface using acollagen gel modulator.

FIG. 10b is an SEM photograph which illustrates the effects ofphotoablation on a laser induced irregular pig corneal surface withoutthe use of a modulator.

FIG. 11 is an SEM photograph which illustrates the effects ofphotoablation on a laser induced irregular human donor corneal surfacewith the use of a collagen gel modulator coating.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and literature references are herebyincorporated by reference in their entirety.

The invention arose from the discovery that collagen compositions areuseful in preparing modulators for use in photoablative procedures forsmoothing irregular corneal surfaces and for protecting adjacent cornealareas surrounding corneal protuberances such as nodules or scars fromundesired photoablation.

As defined herein, the term “biologically compatible” refers to collagenmodified in accordance with the present invention which whenincorporated or implanted into or placed adjacent to the biologicaltissue of a subject, does not induce an immune response or deleterioustissue reaction after such incorporation or implantation or placement;and the term “available cation” refers to metal ions which arereleasably bound to collagen and which may be removed by dialysis.

The type of collagen useful in preparing the collagen modulators of thisinvention is selected from the following groups: purified Type Icollagen, Type IV collagen and Type III collagen, solubilized collagenextracted from intact collagen-rich tissue or a combination of any ofthe foregoing. Preferred as a collagen starting material is purifiedType I collagen derived from animal tissue or predominantly Type Icollagenous product prepared from human tissue. Type I collagen isubiquitous and readily extracted from animal tissues such as dermis andtendon. Type I collagen is the most abundant collagen in connectivetissues, i.e., skin and tendon, and comprises up to 95% of the totalcollagen in skin and tendon. In contrast, type III collagen generallycomprises up to 5% of total collagen in skin. Common sources are bovinetendon and hide and rat tail tendon. Extraction from human tissues isdifficult. U.S. Pat. No. 4,969,912 and U.S. patent application Ser. No.07/572,052, filed Aug. 23, 1990, describe unique methods to disperse andsolubilize human tissue.

A variety of collagen solubilization procedures that are well known inthe art may be used to prepare the modified collagen solutions usefulfor the instant invention. Native collagen is liberated fromnon-collagen connective tissue constituents, e.g. lipids, sugars, andaqueous soluble proteins, extracted from tissue by subjecting it toproteolytic enzymatic treatment by an enzyme other than collagenase.Suitable proteolytic enzymes include pronase and pepsin. The enzymatictreatment removes most of the immunogenic non-helical portions of nativecollagen (telopeptide) and provides a collagen material which is solublein dilute acidic aqueous media. A solution containing the crudesolubilized collagen is then subjected to a series of treatments topurify the soluble type I atelopeptide collagen by separating it frominsoluble collagen, other types of collagen, and non-collagen productsresulting from the proteolytic enzymatic procedure. Conventional methodsfor preparing pure, acid soluble, monomeric collagen solutions bydispersing and solubilizing native collagen are described, for example,in U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037;3,949,073; 4,233,360 and 4,488,911.

It will be understood that any suitable standard procedure may beemployed to prepare a purified collagen solution from any suitablesource so long as the material is undernatured, does not cause anydeleterious effect on the delicate eye tissues, and is capable ofundergoing gelation or fibrillogenesis under physiological conditions. Apreferred method for preparing solubilized collagen solutions for use inpracticing this invention is provided in Example 1 which describespreparation of a purified acid soluble Type I collagen solution frombovine hide, a readily available source of starting material.

The purified acid soluble collagen solution has a concentration, basedon spectrophotometric determination of hydroxyproline content, broadlyranging between about 1 mg and about 20 mg of collagen, preferablybetween about 2 mg and about 5 mg, per ml of solution.

The presence of metal cations in acid solubilized collagen solutions,when adjusted to neutral pH at temperatures of 18° C. to 37° C., mayinitiate molecular interactions leading to premature collagenfibrillogenesis or gel formation, prior to application on a cornealsurface. Hence, in practicing this invention, it is desirable that theacid solubilized collagen be treated to reduce the available cationconcentration prior to and during neutralization. The resultantneutralized acid soluble collagen solution at room temperature orphysiologial temperatures, i.e. 37° C., should be readily applied to thecorneal surface as a liquid coating or film. This coating or film shouldremain in its liquid state and not undergo gelation unless contactedwith an effective amount of a metal cation contained in a physiologicalacceptable solution.

Suitable, but not limiting, methods to reduce levels of available metalcations from solubilized collagen solutions include dialysis,diafiltration, column chromatography using desalting gels, and batchremoval of organic salts using absorbent beads. The preferred method forreducing available metal cation levels present in a solubilized collagensolution is dialysis.

Dialysis is performed at least once against deionized water, with orwithout metal chelating agents, to reduce the available metal cationconcentration to levels which effectively prevent occurrence ofpremature collagen fibrillogenesis or gelation under physiologicalconditions at 37° C. Dialysis reduces the amounts of calcium, magnesium,sodium, potassium and other available metal ions present in the purifiedcollagen solution.

In practicing the invention, it is preferred that the solubilizedcollagen solution be initially dialyzed against an aqueous solutioncontaining metal chelating agents. Suitable, but non-limiting, chelatingagents include aminopolycarboxylic acids such as ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA). Preferred metalchelating dialysis solutions for use in the present invention are EDTAsolutions having a concentration ranging between about 0.025 M and about0.1 M, preferably about 0.03 M.

In the preferred embodiment of the invention, the collagen solution aredialyzed preferably three times against aqueous 0.05 M EDTA solution ata time period of about 24 hours per each dialysis step. The pH of theEDTA solution used in the first dialysis is preferably 4.5. In secondand third dialysis steps, the pH of the EDTA solutions are increased toabout 5.5 and about 7.0, respectively.

Thereafter, the collagen solution is dialyzed at least once againstdeionized water, for a period of about 24 hours, to effectively reducethe amount of metal chelator present in the collagen solution.

Extensive dialysis of EDTA dialyzed collagen solutions against deionizedwater, however, may result in premature gelation during dialysis. Thus,in practicing this invention it is desirable that some EDTA residue beretained in the collagen solution at levels which do not causeirritation to eye tissues. The amount of EDTA retained in the solutionis generally between about 0.02 M and about 0.05 M, preferably betweenabout 0.025 M and about 0.035M.

Upon completion of the dialysis, the dilute neutralized soluble collagensolution may be concentrated, if necessary, to a collagen concentrationbroadly ranging between about 1 and about 10 mg/ml, preferably betweenabout 1 and about 5 mg/ml and most preferably about 3.0 mg/ml, based onspectrophotometric determination of hydroxyproline content. Suitablemethods for concentrating a dilute collagen solution includelyophilization, ultrafiltration, and vacuum drying. A preferred methodfor concentrating the collagen solution is ultrafiltration.

Without being bound to any mechanism or theory of operation in thisinvention, it is believed that treatment of the acid solublized collagensolution with metal chelator-containing solutions (e.g. EDTA disodiumsolutions) and deionized water removes traces of free or unbound metalcations in the collagen solution and/or adjusts the ionic strength ofthe collagen solution which may initiate or promote prematureself-assembly of collagen molecules into fibrils. The collagenself-assembly process is sensitive to a variety of parameters whichinclude ionic strength, pH, temperature, and salt concentration of thesolution. For a discussion, see, K. A. Piez and A. H. Reddi, ed. in“Extracellular Matrix Biochemistry,” Elsevier Publishers; New York, USA,pages 14-28, and Nimni, M. E., ed. in “Collagen: Vol. I Biochemistry,”CRC Press, Inc.; Boca Raton, Fla., USA (1988), pages 1-33 and 113-137.

In one embodiment of the invention, the neutralized soluble collagensolution is used to prepare a modulator, in the form of a gel, on acorneal surface prior to photoablation. The collagen-solution is appliedto the corneal surface in an amount effective to-at least fill insurface depressions and other irregularities. The solution readilyadheres to the surface, filling surface depressions and otherirregularities and provides a smooth surface for subsequentphotoablation. It is preferred that the epithelium on the cornealsurface be removed, e.g. by abrasive scraping or other appropriatetechniques, prior to applying the collagen solution.

The collagen solution may be applied in any suitable manner to at leastfill in surface depressions or irregularities. If desired, the collagensolution may be applied to the corneal surface in amounts to at leastprovide a uniform coating on the corneal surface. Suitable, butnon-limiting, application methods include dropwise addition using asterile pipet or a syringe needle (gauge 16 to gauge 30), brushing andspraying. FIGS. 1 (a-e) illustrate an example of applying a neutralized,soluble collagen solution onto an irregular corneal surface. A flowableneutralized soluble collagen solution is applied (FIG. (1 b)) to anuneven corneal surface (FIG. (1 a)) in an amount to at least fill indepressions or other irregularities (FIG. (1 c)). Thereafter, a metalcation in a physiological medium is applied to the collagen coating(FIG. (1 d)) to convert it to a collagen modulator in a form of a gel(FIG. (1 e)).

Collagen solutions having a concentration greater than 5 mg/ml do notflow easily to form a uniform coating on a surface. In such cases, acontact lens is applied over the solution to assist in forming a uniformlayer of collagen. The application of a contact lens or other suchdevice of known diameter and base curvature that conforms to the shapeof the corneal surface is expected to cause the modulator solution toform a smooth and uniform surface for subsequent ablation. FIGS. 2 (a-g)illustrate the application of a contact lens to form a smoothmodulator-coated surface on the cornea. FIG. (2 a) shows an uneven,irregular corneal surface to which a neutralized solubilized collagensolution is applied (FIG. (2 b)). Thereafter, a contact lens or moldingdevice is placed over the collagen solution (FIG. (2 c)) to uniformlyspread the solution over the corneal surface (FIG. (2 d)). The device isthen removed (FIG. (2 e)) and a metal cation solution is applied (FIG.(2 f)) to the collagen coating which converts it into a modulator gel(FIG. (2 g)).

In practicing the invention, any amount of collagen solution may beapplied to the corneal surface which is effective to at least fill insurface depressions and other irregularities. It will be understood bythe skilled practitioner that the effective amount of collagen solutionemployed is dependent, for instance, on the condition of the cornealsurface, the extent of photoablation required to smooth the surface, andthe concentration of the collagen solution. FIG. (1 f) illustrates across-section of a smooth corneal surface ready for photoablation; thecorneal surface has an adherent collagen modulator coating which atleast fills in depressions and other irregularities on a cornealsurface. FIG. (1 g) illustrates a cross-section of smooth cornealsurface ready for photoablation; the corneal surface has an adherentcollagen modulator as a smooth uniform coating or film on the cornealsurface.

For smoothing of irregular corneal surfaces, the effective amount ofcollagen solution applied is one that at least fills in surfacedepressions and other irregularities. In practicing the invention, amodulator coating having a thickness ranging between about 10 micronsand about 100 microns from the corneal surface, preferably about 50microns, is preferred.

In the case of corneal surfaces having protuberances, e.g. raisedcorneal scars or nodules, the thickness of the modulator coating ispreferably equal to the height of the protrusion. If desired, thecoating may be applied exclusively to areas surrounding the protuberanceto protect these areas from undesired photoablation. FIGS. 3 (a-d)illustrate examples of applying collagen solutions on corneal surfaceshaving a protuberance. FIG. (3 a) illustrates a corneal surface having aprotuberance; FIG. (3 b) show the corneal surface of FIG. (3 a) havingan adherent collagen modulator of approximately the same height as theprotuberance while FIG. (3 c) shows the collagen modulator surroundingthe protuberance and protecting the adjacent areas from undesiredphotoablation. FIG. (3 d) shows a smooth corneal surface followingphotoablation.

Thereafter, the collagen coating is contacted with an amount of a metalcation in a physiologically acceptable solution effective to convert thecollagen coating into a modulator in the form of a gel.

Suitable, but non-limiting, metal cations include sodium, potassium,magnesium and calcium in the form of salts. Suitable but non-limiting,examples of salts include the metal chlorides, bromides, iodides,phosphates, sulfates, acetates. These metal ions are preferablydelivered in a carrier such as a physiologically acceptable solution.e.g water or aqueous buffer solutions. The metal cation solution maycontain one or more types of metal ions. In practicing the invention,the preferred metal cation solutions are phosphate buffered saline orwater solution having a 2.5 M sodium chloride concentration.

The metal cation solutions are applied to the collagen coating in anysuitable manner which does not substantially displace the collagen fromsurface depressions or adversely affect the uniformity of the collagencoating. Application methods include spraying, dropwise addition, andthe like. A preferred method is slowly adding saline solution in adropwise or dribbling fashion to the collagen coating. FIG. (1 d)illustrates an method of applying a cation-containing solution to acollagen-coated corneal surface. Upon formation of collagen gel, excessmetal cation solution may be removed using an appropriate absorbentmaterial, e.g. tissue paper or sponge, prior to photoablation.

Without being bound to any mechanism or theory of operation in thisinvention, it is believed that the cation-exposed solubilized collagenmolecules undergo some degree of self-assembly to form microfibrilswhich may be observed microscopically; a visible “gelation” or gelformation of the collagen molecules occurs. As collagen moleculescontinue to interact, the “gel” becomes more opaque as microfibrils andreconstituted fibrils form. The gelation is believed to be an earlyevent in fibrillogenesis and a phenomenon observed using dilute collagenconcentrations. For a discussion of the fibrillogenesis process, seeNimni, M. E. “Collagen: Vol. I Biochemistry,” CRC Press, Inc.; BocaRaton, Fla., USA (1988), pages 7-16 and Silver, F. H. in “BiologicalMaterials: Structure, Mechanical Properties, and Modeling of SoftTissues”, New York University Press, New York; N.Y. (1987), pages137-163.

In another embodiment of the invention, chemically modifiedpolymerizable soluble collagen solutions are used to prepare collagenmodulatorson a corneal surface. When applied to a corneal surface, thechemically modified collagen adheres to the surface and fills indepressions and other irregularities on the corneal surface. The amountsand methods for applying the modified collagen to a corneal surface aredescribed above. In practicing the invention, it is preferred that theepithelium be removed, by suitable methods, prior to collagenapplication.

Thereafter, the applied modified collagen is subjected to polymerizationconditions, e.g. using short wave UV, to form a thin polymerized filmon-the corneal surface. Short wavelength UV (254 nm) induces freeradical polymerization of aromatic amino acids present in collagen.Chemical initiators of free radical formation, such as sodiumpersulfate, may be optionally added to the collagen coating toaccelerate UV polymerization. The polymerized film firmly adheres to thecorneal surface and is physically removed during the photoablativeprocedure. FIGS. 4 (a-d) illustrate formation of a polymerized modifiedcollagen modulator film on a irregular corneal surface. A flowablemodified collagen solution containing a redox initiator is applied (FIG.(4 b)) to an uneven corneal surface (FIG. (4 a)) in an amount to atleast fill in depressions or other irregularities to form a smoothsurface (FIG. (4 c)). Thereafter, the surface (FIG. (4 d)) is irradiatedwith short wave UV which polymerizes the collagen coating into amodulator film.

A number of modified polymerizable collagen solutions which are usefulin practicing the invention may be used. See, for example, U.S. Pat. No.4,969,912 and co-pending U.S. patent applications Ser. No. 486, 558,filed Feb. 28, 1990; Ser. No. 547, 458, filed Jul. 3, 1990, and Ser. No.646, 944, filed Jan. 29, 1991, which describes modified cross-linkablecollagen solutions and methods for their preparation.

Useful modified collagen compositions which are suitable for use inmaking polymerized modulator films or coatings are based on purifiedsolubilized collagen which are chemically modified with acylatingagents, sulfonating agents, or combinations thereof. Such agents, innon-toxic effective amounts, may be safely employed in preparing amodified collagen modulator.

In practicing this invention, chemically modified collagen is preparedby reacting soluble collagen with at least one acylating agent whichincludes aliphatic, alicyclic and aromatic anhydrides and acid halides.Non-limiting examples of acylating agents include glutaric anhydride,succinic anhydride, lauric anhydride, diglycolic anhydride,methylsuccinic anhydride, methyl glutaric anhydride, dimethyl glutaricanhydride, succinyl chloride, glutaryl chloride, lauryl chloride,phthalic anhydride, methacrylic anhydride, trifluoroacetic anhydride,styrene/maleic anhydride co-polymer, and ethylene/maleic anhydridecopolymer. These chemicals are available from Aldrich Chemical Company(Milwaukee, Wis.). A particularly preferred acylating agent for use inpreparing a modified collagen composition which is suitable for use inthe present invention is glutaric anhydride. An effective amount of anacylating agent is broadly between about 0.5 and about 30 wt. % totalcollagen, preferably between about 2 and about 20 wt. % total collagenin solution.

Useful sulfonating agents for the preparation of modified collagencompositions for use in the present invention include aliphatic,alicyclic and aromatic sulfonic acids or sulfonyl halides. Non-limitingexamples of sulfonating agents for use in preparing collagen modulatorsinclude anthraquinone-1,5-disulfonic acid,2-(chlorosulfonyl)-anthraquinone, 8-hydroxyquinoline sulfonic acid,2-naphthalene- sulfonyl chloride, beta-styrene sulfonyl chloride,2-acrylamido-2-methyl-1-propane sulfonic acid, aniline-2-sulfonic acid,fluorosulfonylbenzene sulfonyl chloride, quinoline sulfonyl chloride andpoly (vinyl) sulfonic acid. These chemicals are also available fromAldrich Chemical Company (Milwaukee, Wis.). Preferred sulfonating agentsare beta-styrene sulfonyl chloride, and quinoline sulfonyl chloride. Aneffective amount of sulfonating agent is broadly between about 0.5 andabout 30 wt. % of the total collagen, preferably between about 2 andabout 20 wt. % of the total collagen in solution.

Non-limiting combinations of acylating agents and/or sulfonating agentsinclude glutaric anhydride/beta-styrene sulfonyl chloride/methacrylicanhydride; glutaric anhydride/ethylene/maleic anhydridecopolymer/methacrylic anhydride; glutaric anhydride/polyvinyl sulfonicacid/methacrylic anhydride; and glutaric anhydride/ethylene/maleicanhydride copolymer/styrene/maleic anhydride copolymer. Preferredcombinations for use in preparing modified collagen modulators includeglutaric anhydride/beta-styrene sulfonyl chloride; glutaricanhydride/phthalic anhydride; and glutaric anhydride/aniline-2-sulfonicacid.

When combinations of two or more acylating agents, sulfonating agents,or mixtures of both agents are used for preparation of modified collagencomposition, the total amount of chemical modifiers is preferablybetween about 2 and about 20 wt. % of collagen in solution.

Modification of collagen is carried out at alkaline pH, in a rangebetween about 7.5 and about 10.0, preferably between about 8.5 and about9.5, and most preferably at about pH 9.0. The acylation reaction may bemonitored more accurately by grossly observing a decrease in pH. Thereaction is terminated when the pH value remains stable at between about5 and about 8, preferably between about 6.5 and about 7.5. The reactionmay also be monitored by removing aliquots and measuring the free amineconcentration of the modified collagen solution as compared to thestarting solution of collagen.

The modification reaction should be complete in between about 5 andabout 90 minutes, preferably between about 20 and about 40 minutes. Thereactions should be carried out at temperatures between about 0° C. andabout 37° C., preferably between about 4° C. and about 25° C.

The reaction may be stopped by adjusting the pH to about 12.0 for about2 minutes. This destroys the residual, unreacted chemical modifier. Themodified collagen is then precipitated by reducing the pH usinghydrochloric acid, acetic acid, nitric acid, sulfuric acid, or otheracid.

The amount of acid must be sufficient to precipitate out the chemicallymodified collagen. Generally precipitation occurs at a pH between about3.5 and about 6.0, preferably between about 4.0 and about 5.0.

The precipitate of reacted collagen which now contains substituentgroups reacted with amine groups (primarily epsilon-amino groups), isrecovered from the mixture using conventional techniques such ascentrifugation or filtration. Centrifugation at between about 3,000 andabout 15,000 rpm for between about 20 and about 60 minutes, preferablybetween about 4,000 and about 12,000, for between about 20 and about 30minutes provides efficient recovery of the precipitate.

After recovery, the precipitate is washed with deionized water andsubsequently dissolved in a physiological solution, e.g., phosphatebuffer (0.1 M) at about pH 7.2. It may be necessary to adjust the pHbetween about 7.0 and about 7.5. This may be done, for example, by theaddition of sodium hydroxide solution.

Following dissolution of the precipitate, the solution is generallyfiltered by conventional filtering means, i.e. a 5 micron filter, andthen centrifuged to remove air bubbles. At this point, the resultingsolution containing chemically modified collagen molecules andaggregates exhibits a viscous consistency, varying degrees oftransparency and clarity, and a characteristic refractive indexdepending on the choice of chemical modifiers, the extent of acylationand on the state of solubility of the starting collagen material.

The chemically modified soluble collagen composition has a collagenprotein concentration broadly ranging between about 5 and about 100 mgper ml of solution, preferably between about 5 and about 50 mg per mland most preferably between about 5 and about 10 mg per ml.

The viscosity of the modified collagen solution, as determined at atemperature of about 25° C., broadly ranges between about 3,000centipoise and about 30,000 centipoise, preferably between about 3,000and about 75,000 centipoise and most preferably about 5,000 centipoise.Viscosity of the solution may be adjusted by the addition of buffer orcollagen precipitate.

The polymerization or crosslinking of the modified collagen compositionsmay be carried out by simply exposing the material to short wave UV(e.g. 254 nm) and atmospheric oxygen. However, the rate ofpolymerization is not practical for use during the excimer laserprocedures. The rate of polymerization may be dramatically increased byadding appropriate redox initiators to the collagen composition followedby exposure to short wavelength UV irradiation. Without such aninitiator, UV polymerization, even in the absence of oxygen, requires atleast 10 minutes.

Prior to polymerization of the modified collagen solution, a suitableinitiator is added to the solution either prior to or after applicationof the modified collagen composition onto the corneal surface. Inpracticing this invention, it is preferred that the initiator be addedto the collagen composition prior to its-application onto the cornealsurface.

Suitable, but non-limiting, examples of initiators include sodiumpersulfate, sodium thiosulfate, ferrous chloride tetrahydrate, sodiumbisulfite and oxidative enzymes such as peroxidase or catechol oxidase.A suitable dosage of the chemical initiator is one that sufficientlypromotes polymerization of the modified collagen within between about 30seconds and about 2 minutes, preferably between about 30 seconds andabout 1 minute, but insufficient to cause oxidative damage to cornealtissue. In practicing this invention, it is preferred that the amount ofinitiator is generally between about 0.5 and about 5 wt. % preferablyless than about 1 wt. % based on total collagen concentration to reducetissue oxidation.

Polymerization by UV irradiation may be accomplished in the short wavelength range by using a standard 254 nm source or UV laser sources. Witha standard 254 nm source of between about 4 and about 12 watts,polymerization generally occurs in between about 30 seconds and abouttwo minutes, preferably no longer than 1 minute, at an exposure distanceof between about 2.5 and about 10 cm, preferably between about 2.5 andabout 5 cm distance. Because excess UV exposure will begin todepolymerize the collagen polymers and cause eye damage, it is importantto limit UV irradiation for short periods. At 254 nm, the penetrationdepth is very limited.

In yet another embodiment of the invention, a glutaricanhydride-modified collagen modulator is provided which undergoestemperature-dependent sol/gel transformations. This extensively modifiedcollagen is preparable by reacting acid soluble collagen having acollagen protein concentration ranging between about 1 and about 10 mgper ml solution with glutaric anhydride in an amount ranging betweenabout 20 and about 30 wt. % total collagen. The chemical modificationreaction conditions and reconstitution of the modified collagen are thesame as described above. The reconstituted glutaric collagen solutionfor use in making the collagen modulator has a collagen concentrationbroadly ranging between about 5 and about 100 mg per ml solution,preferably between about 5 and about 50 mg per ml solution and mostpreferably about 10 mg per ml.

The glutaric collagen melts or liquifies at physiological temperature,e.g 37° C., yet rapidly forms a gel at room temperature without additionof cation solution or induced polymerization. In practicing theinvention, the glutaric collagen is first preheated to aboutphysiological temperature prior to application to the corneal surface.Thereafter, the glutaric collagen coating cools to room temperature andundergoes gelation co form a smooth modulator gel surface ready forphotoablation.

Corneal surfaces having an adherent collagen modulator, as a gel orpolymerized film, are then subjected to the standard procedures used inphotoalblation. The laser sources employed in the photoablationprocedure are preferably non-thermal lasers such as an ultraviolet-orexcimer laser. However, thermal or infra-red lasers may also be used.The ultraviolet lasers are currently preferred as they provide precisebeams of energy which break apart protein bonds, ablating or vaporizingthe cornea as opposed to burning the cornea as caused by thermal lasers.Of course, a wide variety of lasers or other radiation sources may beprovided within the spirit and scope of the invention.

Particularly preferred are ultraviolet lasers such as the excimer typeof far-ultraviolet laser. Such lasers, charged with argon-fluoride gas,have been shown to precisely ablate corneal tissue at wavelengths of 193nm. The laser output of such a laser may be pulsed with typical pulseenergies of more than 300 m³ at a repetition rate of as much as 400pulses per second. Alternatively, radiation from a frequency doubled orquadrupled Nd:YAG laser may be employed giving frequencies in theultraviolet range.

FIGS. 5 (a-g) illustrates post-ablated surfaces, with or without thepresence of a collagen gel modulator. When a collagen modulator isemployed in smoothing operations, a smooth ablated corneal surface(FIGS. 5d-g) is obtained relative to a control surface (FIGS. 5a-c)having no modulator thereon. Application of an eximer laser (FIG. (5 b)to an irregular corneal surface (no modulator present) produced aflattened cornea having irregularities. In contrast, application of aneximer laser (FIG. 5(f)) to an irregular corneal surface (FIG. 5d)having a collagen modulator (FIG. (5 e) resulted in a smooth cornealsurface (FIG. (5 g)).

Upon completion of the photoablation procedure, any remaining residualneutralized soluble or modified collagen modulator gel may be readilyremoved from the corneal surface. Suitable methods for removing the gelinclude spraying with water or a physiological acceptable buffer such assaline solutions. Collagen modulators in the form of a polymerized film,however, are physically removed by the photoablation process.

Methods for performing therapeutic excimer laser keratectomy have beendescribed extensively in the medical literature. It is anticipated thatnumerous improvements or refinements in therapeutic laser photoablationwill be made in the future. The application of the collagen modulatorsof invention in the improved techniques is within the spirit and scopeof the invention.

In addition to its application as an adjunct in therapeuticphotoablation, it is anticipated that collagen modulators would bebroadly useful as removable surface mask to protect surfaces fromundesired exposure to laser sources and to modulate the ablative effectof the laser. For example, the. collagen modulators may be used as anadjunct in photographic and lithographic processes and in circuit boardmanufacture.

The examples set forth below are intended to illustrate the inventionwithout limiting its scope.

EXAMPLE 1 Preparation of Neutralized Acid Soluble Type I CollagenSolution

Fibrous Type I collagen was extracted from bovine material (calf hide)using the following procedure:

Clean, dehaired split hides were purchased from the Andre ManufacturingCo. (Newark, N.J.) and frozen until ready for use. Approximately 200 gof calf hide were thawed at room temperature and cut into approximately1 cm³ pieces using a scalpel and tweezers. The weight of the wet tissuewas recorded. The calf hide was then placed into 15 liters of 0.5 Macetic acid and stirred with a lightening mixer at room temperature forat least one hour. A 10 ml solution of 0.5 M acetic acid containing 2%w/w (or 3.9 g) pepsin from porcine mucosa (Sigma Chemicals, St. Louis,Miss.) was added to the calf hide solution. This solution was stirredovernight with a lightening mixer at room temperature. An additional 10ml 0.5 M acetic acid solution containing 1% w/w (or 1.96 g) pepsin wasadded to the calf hide mixture. The solution was again stirred overnightwith a lightening mixer at room temperature. The dissolved calf hidesolution was refrigerated overnight until a uniform temperature of 4° C.was reached. The pH of the solution was adjusted to 9.0 with 10 N NaOHto denature pepsin. Stirring was maintained throughout the pH adjustmentprocess with a lightening mixer. As collagen will precipitate out at pH9.0 when the temperature is above 6° C., ice cubes were added directlyto maintain the 4° C. temperature. The solution is then refrigerated forat least four hours and then centrifuged at 4° C. for 30 minutes at 9000rpm. The resulting pellet, containing pepsin, was discarded. Thesupernatant, containing collagen, was subjected to a series ofpurification steps.

The collagen solution was subjected to a diafiltration process to removeresidual pepsin and low molecular weight components. An Amicon ModelDC10L/DC10LA ultrafiltration system with a spiral membrane cartridge(SY0100) with a 100,000 kD molecular weight cut off was used.

Thereafter, collagen was precipitated out by adding solid NaCl to thesupernatant to give a final NaCl concentration of 2.5 M. The solutionwas stirred at room temperature for at least two hours. The collagenprecipitate was collected by centrifugation of the solution for 30minutes at 9000 rpm and redissolved in 15 liters of 0.5 M acetic acid, aprocess requiring at least 2 hours. Collagen was reprecipitated outagain by addition of solid NaCl to the solution to a final concentrationof 0.8 M. The solution was stirred for at least two hours and thecollagen collected by centrifugation of the solution for 30 minutes at9000 rpm. This redissolving/precipitation procedure was repeated oncemore. The final pellet, containing purified collagen, was dissolved in0.1 M acetic acid of sufficient volume to provide approximately 0.3 %w/w collagen Type I solution of pH 3.0. The collagen solution was thenfiltered through a 0.45 micron filter to remove particulate matter andsterilized through a 0.22 micron filter. The acid solubilized collagensolution contains a collagen concentration of about 3.0 mg/ml and acation concentration as follows: calcium (0 mg/L), magnesium (0 mg/L),potassium (6.86 mg/L), and sodium (376 mg/L).

FIG. 6 shows that when undialyzed, concentrated solubilized collagensolution (3.0 mg/ml) is adjusted to neutral pH (pH 7.2 using 2.5 NNaOH), fibrillogenesis occurred at 25° C. The absorbance was monitoredat 313 nm for a period of time up to 1 hour.

The acid solubilized collagen solution was then dialyzed against 0.05 MEDTA (99+% A.C.S., disodium salt dihydrate, Aldrich Chemical Co.,Milwaukee, Wis., USA) at a ratio of 1 volume of collagen per 40 volumesof dialyzing solution. Dialysis was conducted using Spectra/Por 1dialysis membrane tubing with a molecular weight cutoff of 6,000-8,000(Spectrum Medical Industries, Inc., Los Angeles, Calif., USA). Threedialysis steps were conducted each for 24 hours and at a temperature of25° C. The collagen solution was then dialyzed two more times, againstfresh EDTA solution with pH adjustments to 5.5 and then to 7.0.Thereafter, the collagen solution was dialyzed against deionized waterfor 24 hours. The resulting neutralized acid soluble solution (50 ml)had a pH of 6.8, a collagen protein concentration of about 3.0 mg/ml,and a cation concentration as follows: calcium (0 mg/L), magnesium (0mg/L), potassium (2.35 mg/L), and sodium (6900 mg/L). The solution wasfiltered through a sterile 0.2 micron filter (Millipore, Bedford, Mass.,U.S.A.).

Aliquots of the dialyzed neutralized collagen solution were concentratedusing two methods: (1) placing 10 ml in a sterile petri dish in theSterile-guard laminar flow hood at 24° C. for 18 hours and (2) placing10 ml of the dialyzed collagen solution in an Amicon stirred cell systemcontaining a 100,000 MW cutoff filter (Amicon, Beverly, Mass., USA).About 40 pounds of pressure were applied using a nitrogen source untilthe collagen concentration was increased to about 5.5 mg/ml. Thematerial, taken from both concentration methods, did not undergo gelatinor fibrillogenesis when incubated at 37° C. for 1 hour.

To evaluate gelation, a 0.1 ml volume of each of the above preparationswere placed on a teflon surface and irrigated with about 0.3 ml of 3 Msodium chloride solution. The collagen immediately formed a firm gelwhich could be removed from the teflon surface using a spatula. The gelwas clear and transparent. After incubation at 37° C. for 10 minutes,the gel became slightly opaque due to fibrillogenesis.

FIG. 7 shows the effect of adding 0.5 ml of 2.5 M saline solution (pH7.0) to 0.5 ml of the neutralized acid soluble collagen solution. Uponaddition of saline, the collagen solution rapidly converted into a gelas evidenced by the sharp increase in absorbance (313 nm).

EXAMPLE 2 Preparation of Glutaric Anhydride Collacen Solution

In this Example, a pure, acid solubilized collagen was prepared asdescribed in Example 1 without the subsequent dialysis steps. Thecollagen solution (3.0 mg/ml) was diluted to 2.8 mg/ml with 0.1 M aceticacid and filtered through a sterile 0.2 micron filter. The solution (100ml) was adjusted to pH 9 and 7 mg (2.5 wt. %) of glutaric anhydride wasadded. The modification reaction proceeded for 20 minutes at 25° C.Thereafter, the pH was decreased to pH 4.3 to precipitate out themodified collagen. The precipitate was recovered by centrifugation andwas washed three times with deionized water. The washed precipitate wasvery fine and granular. This material was dissolved in 10 ml ofphosphate buffer (4 mM, pH 7.8) and the resultant solution was adjustedto pH 7.4 with 1 N NaOH. Thereafter, the solution was filtered through asterile 5 micron filter unit. The modified collagen solution wasviscous, clear, and transparent. The collagen concentration was about 25mg/ml.

EXAMPLE 3 Preparation of an Extensively Modified Glutaric AnhydrideCollagen Solution

In this Example, a pure, acid solubilized collagen was prepared asdescribed in Example 1 without the subsequent dialysis steps; Thecollagen solution (3.0 mg/ml) was diluted to 2.8 mg/ml-with 0.1 M aceticacid and filtered through a sterile 0.2 micron filter. The solution (100ml) was adjusted to pH 9 and 56 mg, (20 wt. %) of glutaric anhydride wasadded. The modification reaction proceeded for 20 minutes at 25° C.Thereafter, the pH was decreased to pH 4.3 to precipitate out themodified collagen. The precipitate was recovered by centrifugation andwas washed three times with deionized water. The washed precipitate wasvery fine and granular. This material was dissolved in 10 ml ofphosphate buffer (4 nM, pH 7.8) and the resultant solution was adjustedto pH 7.4 with 1 N NaOH. Thereafter, the solution was filtered through asterile 5 micron filter unit. The modified collagen solution wasviscous, clear, and transparent. The collagen concentration was about 30mg/ml.

EXAMPLE 4 Preparation of Quinolone Collagen Solution

In this Example, a pure, acid solubilized collagen was prepared asdescribed in Example 1 without the subsequent dialysis steps. Thecollagen solution, at a concentration of 2.2 mg/ml in 0.1 M acetic acid,was filtered through a sterile 0.22 micron filter. The solution (about200 ml) was adjusted to pH 9 with 10 M NaOH and 1 M NaOH and 40 mg (9wt. % based on total collagen) of quinoline sulfonyl chloride was addedas a solid. The modification reaction proceeded for 60 minutes at 25° C.while maintaining the pH at about 9.0. Thereafter, the pH of thesolution was reduced to pH 7 and the solution was filtered sequentiallythrough a 0.45 micron filter and 2.2 micron filter. The filteredmodified collagen solution was precipitated by reducing the pH to 4.6using 6 N HCl and 1 N HC1. The precipitate was recovered bycentrifugation and washed three times with sterile deionized water. Thefinal precipitate was dissolved in phosphate buffered glycerol (4 mMphosphate buffer, 2.2% glycerol, pH 7.8) and adusted to pH 7.2 using 1 NNaOH. The modified collagen solution was filtered through a 5 micronfilter and deaerated by centrifugation at 3500 rpm (IEC Model HN-F2(DAMON/IEC Division, Needham, Mass., USA). The collagen material, at aconcentration of about 5 mg/ml, was clear, viscous, and transparent.

EXAMPLE 5 Preliminary Evaluation of Neutralized Acid Soluble Collagen asModulators

Samples of neutralized acid soluble collagen solution (5.5 mg/ml),prepared in accordance with Example 1, and saline solution at about 3.25M were evaluated using a Tauntan LV 2000 eximer laser (VISX, Sunnyvale,Calif., USA) having a computer controlled module and interactive menu.This laser uses a mixture of argon-fluoride gas to produce a 193 nmwavelength output of 10 Hz and was adjusted to deliver a fluence at thetest surface of 100 to 120 mJ/square cm. The parameters were as follows:energy fluence of 120 mj/cm²; pulse repetition rate 10 Hz. A total of200 pulses were applied. Lucite was used to calibrate the eximer lasersystem. Depressions of 100 microns depth and 5.6 mm diameter were madein a lucite template.

The collagen solution (100 μl) was then applied to the depressions andsmoothed using a glass slide. Saline solution (3.25 M) was flooded ontothe collagen immediately forming a clear gel. The lucite and collagenwere then exposed to eximer laser energy to ablate the surface. Theablated area was 5.6 mm in diameter, and the depth of ablation remainedat 100 microns indicating that the collagen ablated at the same rate asthe lucite.

EXAMPLE 6 Ablation of Enucleated Porcine Cornea Using The CollagenModulator Solution

In this Example, collagen modulators were evaluated in porcine corneausing the Taunton Technologies Model LV 2000 excimer laser andparameters described in Example 5.

Enucleated porcine eyes were obtained from a local packing house. Theeyes were placed in cold sterile saline solution and refrigerated priorto use. Before ablation experiments, the epithelium on the cornealsurface was removed using a scalpel blade. An eye was placed in a holderand properly aligned under the eximer laser.

After 600 pulses, an ablation spot of 5.6 mm was formed on the cornealsurface at a depth of between 50 and 100 microns (FIG., 8 a).

A 0.1 ml aliquot of a neutralized acid soluble collagen (3.0 mg/ml),prepared in accordance with Example 1, was then applied, using a pasteurpipet, to the deepithelized corneal surface of a second eye. Anequivolumne of 3.25 M saline solution was then added, with a pasteurpipet, to the collagen coating. The coating instantly converted into agel. The photoablation procedure, as described above, was thenperformed. When a collagen gel modulator was formed on the surface ofthe porcine cornea, the depth of the ablation spot was reduced by about75%. The ablation depth was less than 25 microns compared to 100 micronsfor controls with no collagen (FIG. 8b).

In a separate experiment, crude defects were formed on two porcinecorneas using a No. 64 surgical blade. When ablated without a collagenmodulator (control eye), using the conditions noted above, the cornealsurface became very uneven in the ablation area (FIG. 9a). In contrast,when a collagen modulator was present on the corneal defect, the roughsurface became much smoother and more uniform (FIG. 9b) after ablationthan the untreated control eye (FIG. 9a). This experiment clearlydemonstrated the beneficial effects of using a collagen gel modulator onthe corneal surface in providing a smooth uniform surface.

EXAMPLE 7 Ablation of an Irregular Porcine Cornea Using a NeutralizedAcid Soluble Collagen Modulator Coating

In this Example, a 2 cm×2 cm stainless steel or polymer mesh (0.8mm×0.67 mm) was applied to the surface of a deepithelized pig cornea.Photoablation was initiated and 600 pulses applied using the settingsdescribed in Example 5. Ablation results in an impression of the screenin the corneal surface at a depth of about 50 microns. Neutralized acidsoluble collagen solution (3.0 mg/ml), prepared in accordance withExample 1, was applied to the surface to fill in the depressions formedby the screen, followed by addition of saline solution as described inExample 6. The eye was then subject to an additional 900 pulses. Thecollagen modulator appeared to ablate at the same rate as the cornealtissue preventing deepening of the depressions as the screen impressionwas removed by photoablation (FIG. 10a). When the collagen modulator wasabsent, the impression of the screen remained after ablation (FIG. 10b).Thus, these experiments clearly demonstrated the beneficial effects ofthe collagen modulator in providing an important adjunct tophotoablation keratectomy.

EXAMPLE 8 Ablation of an Irregular Human Cornea Using a Neutralized AcidSoluble Collagen Modulator Coating

In this Example, a human donor eye, rejected for corneal translation,was obtained from the Kentucky Lions Eye Bank. The eye was stored incold sterile saline buffer at 5° C. Before ablation, the epithelium wasremoved using a scalpel blade. The eye was placed in a holder andaligned under the eximer laser. A screen impression was made on thedeepithelized cornea as described in Example 7. A collagen gel modulatorwas formed on the cornea and the corneal surface was reexposed to theeximer laser for an additional 900 pulses, as described in Example 5.The results shown in an SEM photograph (FIG. 11) demonstrate thebeneficial effects of using a collagen modulator in providing a smoothcorneal surface.

EXAMPLE 9 Ablation of an Irregular Porcine Cornea Using a ModifiedCollagen Modulator Film

In this Example, a 2 cm×2 cm stainless steel or polymer mesh (0.8mm×0.67 mm) is applied to the surface of a deepithelized pig cornea.Photoablation is initiated and 600 pulses are applied using the settingsdescribed in Example 5. Ablation results in an impression of the screenin the corneal surface at a depth of about 50 microns. Approximately 100μl of a glutaric anhydride modified collagen solution, prepared inaccordance with Example 2 and containing 5 wt. % sodium persulfate isapplied to the corneal surface with a syringe fitted with a 25 gaugeneedle to fill in the depressions formed by the screen. Thereafter, thecoated corneal surface is subjected to 1.5 minutes of 254 nm UVirradiation at a distance of 4 cm (4 watt portable UV lamp providing 350microwatts/cm² intensity at 3 inches, Ultraviolet Products, Inc., SanGabriel, Calif., U.S.A.) to polymerize the glutaric collagen into a thinfilm. The eye is then subject to an additional 900 pulses as describedin Example 3. The collagen modulator appears to ablate at the same rateas the corneal tissue and prevents deepening of the depressions as thescreen impression is removed by photoablation. When the collagenmodulator is absent, the impression of the screen remains afterablation. Thus, these experiments clearly demonstrate the beneficialeffects of the collagen modulator film in providing an important adjunctto photoablation keratectomy.

EXAMPLE 10: Ablation of an Irregular Porcine Cornea Using an ExtensivelyModified Glutaric Anhydride Collagen Modulator Gel

In this Example, a 2 cm×2 stainless steel or polymer mesh is applied tothe surface of a deepithelialized pig cornea. Photoablation is initiatedand 600 pulses applied using the settings described in Example 5.Ablation results in the formation of an impression of the screen in thecorneal surface at a depth of about 50 microns. 100 ul of meltedglutaric derivatized collagen solution (37° C.), prepared in accordancewith Example 4, is applied to the irregular corneal surface using asyringe fitted with a 25 gauge needle. The collagen modulator forms afirm gel as the collagen equilibrates to room temperature. The eye isthen subjected to 900 pulses as described in Example 5. The collagenmodulator appears to ablate at the same rate as the intact cornea andprevents deepening of the depressions as the screen impression isremoved by photoablation. When the modulator is absent, the impressionof the screen remains, and becomes deeper, after ablation. Thus, theseexperiments clearly demonstrate the beneficial effects of the collagenmodulator film in providing an important adjunct to photoablationkeratectomy.

EXAMPLE 1: Ablation of an Irregular Porcine Cornea Using a QuinolineCollagen Modulator Film

In this Example, a 2 cm×2 stainless steel or polymer mesh is applied tothe surface of a deepithelialized pig cornea. Photoablation is initiatedand 600 pulses applied using the settings described in Example 5.Ablation results in the formation of an impression of the screen in thecorneal surface at a depth of about 50 microns. Approximately 100 ul ofa quinoline sulfonyl chloride modified collagen solution, prepared inaccordance with Example 4 and containing 5 wt. % sodium persulfate isapplied to the corneal surface with a syringe fitted with a 25 gaugeneedle to fill in the depressions formed by the screen. Thereafter, thecoated corneal surface is subjected to 1.5 minutes of 254 nm UVirradiation at a distance of 4 cm (4 watt portable UV lamp providing 350microwatts/cm² intensity at 3 inches, Ultraviolet Products, Inc., SanGabriel, Calif., U.S.A.) to polymerize the glutaric collagen into a thinfilm. The eye is then subject to an additional 900 pulses as describedin Example 5. The collagen modulator appears to ablate at the same rateas the corneal tissue and prevents deepening of the depressions as thescreen impression is removed by photoablation. When the collagenmodulator is absent, the impression of the screen remains afterablation. Thus, these experiments clearly demonstrate the beneficialeffects of the collagen modulator film in providing an important adjunctto photoablation keratectomy.

What is claimed is:
 1. A method for preparing a collagen modulator on acorneal surface, said corneal surface having depressions, whichcomprises: applying a neutralized acid soluble collagen solution ontosaid corneal surface in an amount sufficient to at least fill in saiddepressions; said neutralized acid soluble collagen solution on saidcorneal surface having been contacted with an effective amount of atleast one metal cation, said metal cation contained in a physiologicalacceptable solution, so as to convert said soluble solution to acollagen modulator gel.
 2. The method according to claim 1, wherein saidcollagen solution comprises a Type I collagen solution.
 3. The methodaccording to claim 1, wherein said metal cation is selected from thegroup consisting of sodium, potassium, magnesium, and calcium.
 4. Themethod according to claim 1, wherein said metal cation is present insaid physiologically acceptable solution in a concentration rangingbetween about 0.01 M and about 0.05 M.
 5. The method according to claim1, wherein said collagen solution contains between about 1 mg and about10 mg collagen protein per ml of solution.
 6. The method according toclaim 1, wherein said corneal surface has been deepithelialized.